Dot blot test

Quantification of the DNA isolated from the product involves concurrent inclusion in the dot blot assay of a set of spots, containing known quantities of DNA, and being derived from the producer cell. After autoradiography, the intensity of the test spot is compared with the standards. 

RFLP analysis of the p-globin gene for genetic testing has been replaced, by PCR In combination with ASO probes on dot blots. The blot shown here corresponds to the family whose pedigree is shown in Figure T7-9. In the mutant allele, glutamate (E) at codon 6 is replaced by valine (V). 

When the amplified material was tested under stringent conditions using aUele-specific probes, the results indicated that the child was heterozygous at tiie p-globin locus. Which dot blot shown below best represents the results from this family ... 

For additional discussion of allele-specific oligonucleotide (ASO) probes and dot blots, see Section I, Chapter 7 Genetic Testing. 

There are many ways of testing a gold probe, but the most convincing is by using a known positive sample Therefore, this could be a histological section, an electron microscope specimen, or a dot-blot. Estimation of the concentration of the probe by optical density measurements is a good method to standardize the concentration of probes from one batch to another, but in addition, it is always preferable to test the performance of the probes on known positive samples. 

Hybridization analysis using immobilized DNA includes dot blots and slot blots. Dot and slot blots are named for the circular and slotted well shapes in the templates used to present test samples to a membrane surface. They eliminate the need for restriction digestion and electrophoretic resolution steps by depositing samples of DNA to be tested directly onto a hybridizable membrane surface. Dot blot methodology is faster and easier for hybridization screening than Southern blotting, especially when many samples are to be screened... 

This assay format is well suited to the screening of test compounds for potential antiviral activity. The assay provides a minimal assessment of antiviral activity by measuring the levels of HBV virion release from the cells, as well as providing a measurement of cytotoxicity (see Subheading 3.2.2.), Two rows of cells will be required for each compound, plus four rows for the assay controls (two for untreated, and two for positive antiviral control (e.g., 3TC). After treating for 9 d, the media are harvested from the antiviral plates and transferred to 96-well U-bottomed plates. They are then centrifuged, and supernatant is transferred to tubes for dot-blot hybridization analysis of HBV virion DNA. The medium is aspirated off of the toxicity plates and discarded. Toxicity plates are then incubated with neutral red dye (methylthiouracil [MTT] can also be used if preferred), washed with DPBS, developed with an acetic acid/ ethanol solution, and assayed in a plate reader. 

A similar technique that bypasses the electrophoretic separation step is known as dot blotting. A protein sample to he analyzed is applied to a membrane surface as a small dot and dried. The membrane is then exposed to a labeled antibody specific for the test antigen contained in the dotted protein mixture. After washing, bound labeled antibody is detected with a photometric or chemiluminescent detection system. 

The amplicons prepared are subsequently analyzed by a variety of methods. They can be hybridized to oligonucleotide probes in reverse or forward dot-blot assays. The panel of probes is chosen to cover critical polymorphic positions in the HLA gene tested. The pattern of reactivity of the panel can then be analyzed to assign allele identities. ... 

There are currently several methods for analysis of the amplified target DNA. For HIV-1, liquid hybridization with radioactively labeled probes is used (K12). Tests for HLA genes and sickle cell anemia utilize the reverse dot-blot format with a nylon membrane (S3). Each clinical research format has a well-characterized detection method defining the optimum probe concentration, the hybridization times and temperatures, as well as the concentrations of indicator reagents. Table 5 describes the optima and tolerances of a nonradioactive dot-blot assay that uses biotinylated probes and detection by a chemiluminescent substrate and a strepta-vidin-HRP conjugate. 

When water samples collected from the field were spiked with the 6-endotoxin and tested, sensitivity and precision of the ELISA procedure decreased regardless of the pH tested. Two procedures, anion exchange chromatography and dot-blotting, were explored for sample clean-up and concentration when sample work-up is needed. In addition, problems with assay development are also discussed. As more biochemical agents are introduced into the agricultural market, derived either from classical or molecular approaches, it is critical that analytical methods are in place for quality control and to monitor the presence of these agents in the environment. 

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